Heart disease and stroke are major causes of morbidity and mortality in the United States. Mounting evidence suggests that an episode of cardiac ischemia (heart attack) or cerebral ischemia (stroke) initiate a chain of biochemical events that result in increased intracellular Ca2+ concentration, which in turn activates calpain. Activated calpain degrades structural proteins resulting in cell death. Calpain is therefore considered as an attractive therapeutic target for intervention in heart attack and stroke. The long-term goal is to discover novel calpain inhibitors as treatment for heart attack and stroke. The specific aim is to determine the specificities of the S subsites of calpain with the objective of developing potent and selective inhibitors of the enzyme. The structural requirements for inhibitor binding to the S subsites of calpain has been well investigated. On the contrary, the structural requirements for inhibitor binding to the S? subsites of calpain have only been marginally investigated. This is a significant gap in the literature that must be filled because knowledge about the S? subsite specificities of calpain will provide valuable information that can aid in the design of selective inhibitors of the enzyme. Inhibitors with natural and unnatural D- and L-amino acids at the P1? and P2? positions of the inhibitors will be synthesized to probe the specificities of the S1? and S2? subsites of calpain. Comparative Molecular Field Analysis (CoMFA) will be used to generate a binding site model for the inhibitors and the model will be used to design and predict the calpain inhibitory potency of novel inhibitors before chemical synthesis and enzymology. Potent inhibitors will be tested for their ability to enter cell and inhibit intracellular calpain. Selected potent and cell permeable inhibitors will be tested in the rat isolated heart ischemia model for cardioprotective effect.